New Kunitz-Type HCRG Polypeptides from the Sea Anemone Heteractis crispa.

Irina Gladkikh,Alexis Ivanov,Margarita Monastyrnaya,Valentin Tabakmakher,Oksana Sintsova,Oksana Gnedenko,Kuo-Feng Hua,Emma Kozlovskaya,Elena Zelepuga

doi:10.3390/md13106038

Abstract

Sea anemones are a rich source of Kunitz-type polypeptides that possess not only protease inhibitor activity, but also Kv channels toxicity, analgesic, antihistamine, and anti-inflammatory activities. Two Kunitz-type inhibitors belonging to a new Heteractis crispa RG (HCRG) polypeptide subfamily have been isolated from the sea anemone Heteractis crispa. The amino acid sequences of HCRG1 and HCRG2 identified using the Edman degradation method share up to 95% of their identity with the representatives of the HCGS polypeptide multigene subfamily derived from H. crispa cDNA. Polypeptides are characterized by positively charged Arg at the N-terminus as well as P1 Lys residue at their canonical binding loop, identical to those of bovine pancreatic trypsin inhibitor (BPTI). These polypeptides are shown by our current evidence to be more potent inhibitors of trypsin than the known representatives of the HCGS subfamily with P1Thr. The kinetic and thermodynamic characteristics of the intermolecular interactions between inhibitors and serine proteases were determined by the surface plasmon resonance (SPR) method. Residues functionally important for polypeptide binding to trypsin were revealed using molecular modeling methods. Furthermore, HCRG1 and HCRG2 possess anti-inflammatory activity, reducing tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) secretions, as well as proIL-1β expression in lipopolysaccharide (LPS)-activated macrophages. However, there was no effect on nitric oxide (NO) generation.

Highlights

According to phylogenetic studies, sea anemones are one of the oldest group of venomous marine animals [1]
Genes encoding Kunitz-type polypeptides have evolved from a common ancestor, which is responsible for the serine protease binding not undergoing any significant changes [12]
Gel filtration of polypeptides contained in the water solution of acetone powder on Akrilex P-4 column gave three protein fractions (Figure 1A, peaks 1–3) with biological active polypeptides

Summary

Introduction

Sea anemones (phylum Cnidaria, class Anthozoa) are one of the oldest group of venomous marine animals [1]. Sea anemone venom contains a variety of different proteinaceous toxic components, which are important for prey capture and defending against predators. They remain one of the most poorly studied venomous animal lineages, several sea anemone biologically active polypeptides have been characterized for their structurally and functionally properties. Kunitz-type polypeptides contain one of the most evolutionarily ancient and the most conserved among the protein structural motifs, the Kunitz fold [10,11], which was first found in the bovine pancreatic trypsin inhibitor (BPTI) [13] The representatives of this group form a compact and stable alpha+beta fold stabilized well by three conservatively positioned disulfide bridges with the bonding patterns

Methods

Results

Conclusion